DESCRIPTION(adapted from applicant's abstract): The overall objective of this project is to investigate the efficacy of liver or muscle-directed gene therapy using recombinant adeno-associated virus (rAAV) vectors in the treatment of phenylketonuria (PKU) using, as a model, the phenylalanine hydroxylase (PAH) deficient Pahenu2 mouse. Recombinant adeno-associated virus has in recent studies shown promise as a gene delivery vector for both liver and muscle. We propose to compare the effects of muscle-directed vs. liver-directed rAAV-mediated gene therapy upon hyperphenylalaninemia in the Pahenu2 mouse. Restoration of liver enzyme activity is the most obvious approach for gene therapy of PKU. However, the disease pathophysiology associated with PKU is caused by circulating toxins, namely phenylalanine and its metabolites; PAH deficiency has no direct pathologic effect upon the liver. Therefore, any treatment approach that can effectively remove the toxic metabolites from the body could prevent disease-associated pathology. We have shown previously that PAH expression in muscle can lower serum phenylalanine levels if the enzyme is supplied with sufficient tetrahydrobiopterin (BH4), a required cofactor for PAH activity. However, attaining a sufficient continuous supply through exogenous administration of BH4 is very difficult and expensive. We propose two potential methods for avoiding this complication: engineering of skeletal muscle to express, along with PAH, components of the BH4 synthetic pathway or alternatively to induce muscle expression of phenylalanine ammonia lyase (PAL), an enzyme from yeast that requires no exogenous cofactors. In this project, we will first compare the ability of rAAV vectors to produce stable PAH expression in liver and muscle and assess the effect upon hyperphenyl-alaninemia in Pabenu2 mice. Secondly, we will develop germline modified mice that carry a muscle-specific transgene containing the PAH cDNA and elements of the BH4 synthetic pathway (GTP cyclohydrolase I (GTPCH) or GTPCH and pyruvoyltetrahydropterin synthase (PTPS)) in an attempt to induce both constitutive PAH expression and BH4 synthesis. We will breed these animals to Pahenu2 mice to determine whether muscle PAH expression together with BH4 synthesis can correct hyperphenylalaninemia in PKU. Finally, we will investigate PAL expression induced either by germline modification or rAAV-mediated gene transfer in Pahenu2 mouse muscle, as a possible treatment of hyperphenylalaninemia. The ultimate goal of our research is the development of effective and safe gene therapy for phenylketonuria.